The invention relates to the genetic manipulation of plants, particularly to modulating DNA metabolism in transformed plants and plant cells.
Replication protein A (RPA) is a single-stranded DNA-binding protein that is required for multiple processes in eukaryotic cells. RPA from human cells is a stable complex of 70-, 32-, and 14-kDa subunits. Homologues of RPA have been identified in all eukaryotes examined. However, only human RPA and closely related homologues can support SV40 DNA replication.
The RPA complex appears to be highly conserved in all eukaryotes. The three RPA genes in budding yeast cells are essential for cell viability. Nevertheless, yeast RPA only partially substitutes for human RPA in the in vitro replication of simian virus 40 indicating that species-specific interactions between RPA and other replication proteins may be important for its biological activity.
RPA binds tightly to single stranded DNA as a heterotrimeric complex. The binding activity has been localized to the 70 kDa subunit. The affinity of RPA for both double-stranded DNA and RNA is at least three orders of magnitude lower than it is for single-stranded DNA. It has been reported that RPA binds preferentially to the pyrimidine-rich strand of both S. cerevisiae sequences and the SV40 origin of replication. However, studies examining the determinants of replication origins in S. cerevisiae indicate that this preferential binding is not critical for the initiation of DNA replication.
Subunits of RPA in the 70-, 32- and 14 kDa ranges have been identified from various sources. The 32 kDa subunit has also been referred to as xe2x80x9cRPA2xe2x80x9d, xe2x80x9cBxe2x80x9d, xe2x80x9csmallxe2x80x9d, xe2x80x9c32 kDaxe2x80x9d, xe2x80x9cP32xe2x80x9d, xe2x80x9cP34xe2x80x9d, and xe2x80x9cmiddlexe2x80x9d subunit. For the purposes of this invention, the xe2x80x9cmiddlexe2x80x9d subunit is intended as the subunit having a molecular weight of about 32 kDa.
The middle subunit of RPA has a role in cell cycle regulation; single stranded DNA binding; affinity of DNA binding; species-specificity of DNA binding; DNA recombination, repair, replication and metabolism; and response to DNA damages. (Anderson (1966) Calif. Inst. Technol.; Seroussi et al. (1993) J. Biol. Chem. 268:7147-54; Kenny et al. (1989) Proc. Natl. Acad. Sci. USA 86:9757-61; Brush et al. (1995) Methods Enzymol. 262:522-48, Stigger et al. (1994) Proc. Natl. Acad. Sci. USA 91:579-83; Philipova et al. (1996) Genes Dev. 10:2222-33).
Much research has centered on the exploration of the biochemical and genetic mechanisms by which cell cycle regulation of DNA synthesis is achieved. While there have been advances in delineating the existence of cell cycle proteins, more information is needed on the mechanism of action of DNA replication, recombination, and repair. Furthermore, methods for regulating or altering the cell cycle is needed.
Related Literature
Braun et al. (1997) Biochemistry 36:8443-8454; report on the role of protein-protein interactions and the function of replication protein A. It is reported that RPA modulates the activity of DNA polymerase xcex1 by multiple mechanisms.
Loor et al. (1997) Nucleic Acids Research 25:5041-5046 report on the identification of DNA replication in cell cycle proteins that interact with proliferating cell nuclear antigen.
Longhese et al. (1994) Molecular and Cellular Biology 14:7884-7890 report that replication factor A is required for in vivo DNA replication, repair, and recombination.
Stigger et al. (1998) J. Biol. Chem. 273:9337-9343 provide a functional analysis of human replication protein A in nucleotide excision repair.
Abremova et al. (1997) Proc. Natl. Acad. Sci. USA 94:7186-7191 report that the interaction between replication protein A and p53 is disrupted after ultraviolet damage in a DNA repair-dependent manner.
New et al. (1998) Nature 391:407-410 reports that RAD52 protein stimulates DNA strand exchange by RAD51 and replication protein A. Stimulation was dependent on the concerted action of both RAD51 protein and RPA implying that specific protein-protein interactions between RAD52 protein, RAD51 protein and RPA are required.
Dutta et al. (1992) EMBO J 11(6):2189-2199 and Niu et al. (1997) J. Biol. Chem. 272(19):12634-41 report cell cycle-dependent phosphorylation of the middle subunit of RPA, implying a role for the subunit in cell cycle regulation.
Bochkareva et al. (1998) J. Biol. Chem. 273(7):3932-3936 report the formation of a single stranded DNA binding site on the human RPA middle subunit.
Mass et al. (1998) Mol. Cell. Biol. 18(11):6399-6407 report that the RPA middle subunit contacts nascent simian virus 40 DNA, particularly the early DNA chain intermediates synthesized by DNA polymerase alpha-primase (RNA-DNA primers), but not more advanced products.
Lavrik et al. (1998) Nucleic Acids Res 26(2):602-607 report on location of binding of individual subunits of human RPA to DNA primer-template complexes in various elongation reactions.
Sibenaller et al. (1998) 37(36):12496-12506 report that differences in the activity of the middle (32 kDa) and the small (14 Kda) subunits of RPA are responsible for variations in the single stranded DNA-binding properties of sacchromyces cerevisiae and human RPA, thus implying a role for the subunits in species-specificity of DNA binding of RPA.
Compositions and methods for modulating DNA metabolism in a host cell is provided. Particularly, the complete cDNA and amino acid sequence for homologues of maize replication protein A (RPA) large- and middle subunits are provided. The sequences of the invention find use in modulating DNA replication, DNA repair, and recombination.
Transformed plants can be obtained having altered metabolic states. The invention has implications in genetic transformation and gene targeting in plants. Additionally, the methods can be used to promote cell death particularly in an inducible or tissue-preferred manner.